Yong Hai Nan

Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides.

To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH(2) (X indicates Leu or Lys/Arg), was synthesized. K(6)L(2)W(3) displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K(8)W(3), K(7)LW(3) and K(6)L(2)W(3)) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R(8)W(3), R(7)LW(3) and R(6)L(2)W(3)), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K(6)L(2)W(3)-D and R(6)L(2)W(3)-D) and Orn/Nle-containing peptides (O(6)L(2)W(3) and O(6)L(2)W(3)) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K(6)L(2)W(3) and R(6)L(2)W(3)), highlights the clinical potential of such peptides. K(6)L(2)W(3), R(6)L(2)W(3), K(6)L(2)W(3)-D and R(6)L(2)W(3)-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K(6)L(2)W(3), K(5)L(3)W(3), R(6)L(2)W(3), O(6)L(2)W(3), O(6)L(2)W(3), and K(6)L(2)W(3)-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents.

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37.

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1.

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro(8) for Gly(8) in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly(8) of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro(8) whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys(8). Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].

Design of novel indolicidin-derived antimicrobial peptides with enhanced cell specificity and potent anti-inflammatory activity

Indolicidin (IN) is a 13-residue Trp-rich antimicrobial peptide isolated from bovine neutrophils. To develop novel IN-derived antimicrobial peptides with enhanced cell specificity (therapeutic index) and potent anti-inflammatory activity, several IN analogs were synthesized by Pro-->Lys substitution. All IN analogs displayed an increase in therapeutic index by 3- to 15-fold relative to parental IN. IN and its analogs induced a significant membrane depolarization against intact Staphylococcus aureus in a dose-dependent manner and depolarized membrane potential at 5 microg/ml (MIC for S. aureus) almost completely. However, these peptides caused less than 40% calcein leakage from negatively charged EYPG/EYPE liposomes mimicking bacterial membranes at 10 microg/ml. Based on these results, we hypothesize that IN and its analogs kill microorganisms via the formation of small ion channels that permit transit of ions or protons, but not molecules as large as calcein. Furthermore, IN and its analogs induced a remarkable suppression in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression in LPS-stimulated mouse macrophage RAW264.7 cells. All IN analogs showed LPS-binding activity comparable to that of IN. Taken together, their potent antimicrobial, anti-inflammatory and LPS-neutralizing activities similar to those of IN, coupled with their no cytotoxicity, our designed IN analogs make excellent candidates for novel antimicrobial and anti-sepsis agents.

Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich α-helical model antimicrobial peptide and its diastereomeric peptides

To investigate the effect of the number and distribution of d-amino acids introduced into non-cell-selective alpha-helical antimicrobial peptides on the cell selectivity, protease stability and anti-inflammatory activity, we synthesized an 18-meric Leu/Lys-rich alpha-helical model peptide (K(9)L(8)W) and d-amino acid-containing diastereomeric peptides. Increasing in cell selectivity of the peptides was increased in parallel with increasing in the number of d-amino acids introduced. Despite having the same number of d-amino acids, D(9)-K(9)L(8)W-1 had better cell selectivity than D(9)-K(9)L(8)W-2, indicating that a dispersed distribution of d-amino acids in diastereomeric peptides is more effective for cell selectivity than their segregated distribution. D(3)-K(9)L(8)W-2, D(6)-K(9)L(8)W, D(9)-K(9)L(8)W-1 and D(9)-K(9)L(8)W-2 showed complete resistance to tryptic digestion. Furthermore, K(9)L(8)W and all of its diastereomeric peptides significantly inhibited nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA expression and tumor necrosis factor-alpha (TNF-alpha) release in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells at a lower concentration than bactericidal concentration. The order of anti-inflammatory activity for the peptides was K(9)L(8)W approximately D(3)-K(9)L(8)W-1 approximately D(3)-K(9)L(8)W-2 approximately D(6)-K(9)L(8)W approximately D(9)-K(9)L(8)W-2>D(4)-K(9)L(8)W>D(9)-K(9)L(8)W-1. Increasing in hydrophobicity or alpha-helicity of the peptides was more closely correlated with increasing in hemolytic activity and anti-inflammatory activity than antimicrobial and LPS-disaggregation activities. Collectively, we successfully developed several d-amino acid-containing antimicrobial peptides (D(4)-K(9)L(8)W, D(6)-K(9)L(8)W and D(9)-K(9)L(8)W-1) with good cell selectivity, protease stability and potent anti-inflammatory activity. These antimicrobial peptides could serve as templates for the development of peptide antibiotics for the treatment of sepsis, as well as microbial infection.

Cell selectivity, mechanism of action and LPS-neutralizing activity of bovine myeloid antimicrobial peptide-18 (BMAP-18) and its analogs.

To develop novel antimicrobial peptides (AMPs) with improved cell selectivity and potent LPS-neutralizing activity, we synthesized an 18 N-terminal residues peptide (BAMP-18) of bovine myeloid antimicrobial peptide-27 (BMAP-27) and its analogs (BMAP-18-W, BMAP-18-L, BMAP-18-I and BMAP-18-f). BMAP-18 and its analogs displayed much higher cell selectivity (about 4-97-fold increased) as compared to parental BMAP-27 because of their decreased hemolytic activity and retained antimicrobial activity. BMAP-27 caused near-complete dye leakage from bacterial-membrane-mimicking vesicles even at very low concentration of 0.5μM, whereas BMAP-18 and its analogs induced very little dye leakage (less than 40%) even at 16μM. These peptides induced near-complete membrane depolarization of Staphylococcus aureus cells under their MIC (4μM). These results suggests that BMAP-18 and its analogs exhibit lethality toward microbes due to their ability to form small channels that permit the transit of ions or protons, but not molecules as large as calcein, and not by the membrane-disruption/perturbation mode. BMAP-18 and its analogs significantly inhibited nitric oxide (NO) production or tumor necrosis factor-α (TNF-α) release in LPS-stimulated mouse macrophage RAW264.7 cells at 10μM. In particular, BMAP-18-W showed LPS-neutralizing activity comparable to that of BMAP-27. There was a significant linear correlation between the increase in the hydrophobicity of peptides and LPS-neutralizing activity. Although BMAP-18-W has lower hydrophobicity than BMAP-18-L, it showed higher LPS-neutralizing activity as compared to BMAP-18-L. This result suggests other important parameters of AMPs may be involved in their LPS-neutralizing activity, as well as positive charge and hydrophobicity.

Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin

To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp-->Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50-90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5-10 microg mL(-1)). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 microg mL(-1). Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.

The thin line between cell-penetrating and antimicrobial peptides: the case of Pep-1 and Pep-1-K ‡

Cell‐penetrating peptides (CPPs) are cationic oligopeptides able to translocate across biological membranes without perturbing them, while antimicrobial peptides (AMPs) kill bacteria mainly by disrupting their membranes. The two peptide classes share several characteristics (charge, amphipathicity, helicity, and length), and therefore the molecular properties discriminating between the two different bioactivities are not clear. Pep‐1‐K (KKTWWKTWWTKWSQPKKKRKV) is a new AMP derived from the widely studied CPP Pep‐1 (KETWWETWWTEWSQPKKKRKV), or ‘Chariot’, known for its ability to carry large cargoes across biological membranes. Pep‐1‐K was obtained from Pep‐1 by substituting the three Glu residues with Lys, to increase its cationic character. Previous studies showed that these modifications endow Pep‐1‐K with a potent antimicrobial activity, with MICs in the low micromolar range. Here, we characterized the interaction of Pep‐1 and Pep‐1‐K with model membranes to understand the reason for the antimicrobial activity of Pep‐1‐K. The data show that this peptide causes vesicle aggregation, perturbs membrane order, and induces the leakage of ions, but not of larger solutes, while these effects were not observed for Pep‐1. These differences are likely due, at least in part, to the higher affinity of Pep‐1‐K toward anionic bilayers, which mimick the composition of bacterial membranes. Copyright

Antimicrobial activity, bactericidal mechanism and LPS-neutralizing activity of the cell-penetrating peptide pVEC and its analogs.

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright

Mammalian cell toxicity and candidacidal mechanism of Arg- or Lys-containing Trp-rich model antimicrobial peptides and their d-enantiomeric peptides.

In this study, we investigated the mammalian cell toxicity and candidacidal mechanism of Arg- or Lys-containing Trp-rich model antimicrobial peptides (K(6)L(2)W(3) and R(6)L(2)W(3)) and their d-enantiomeric peptides (K(6)L(2)W(3)-d and R(6)L(2)W(3)-d). Arg-containing peptides were more toxic to human erythrocytes and mammalian cells as compared to Lys-containing peptides. Arg-containing peptides is slightly more hydrophobic than Lys-containing counterparts, as judged from their reverse phase-high performance liquid chromatography (RP-HPLC) retention time. These results suggested that a little difference in hydrophobicity of these peptides affect their hemolytic activity and mammalian cell toxicity. Interestingly, K(6)L(2)W(3) and K(6)L(2)W(3)-d almost similar mammalian cell cytotoxicity, whereas R(6)L(2)W(3)-d showed much higher cytotoxicity as compared to R(6)L(2)W(3). A low ability to facilitate fluorescent marker escape from Candida albicans membrane-mimicking vesicles suggested that the major target site of Lys-containing peptides may be not the cell membrane but the cytoplasm of C. albicans. Confocal laser-scanning microscopy revealed that FITC-labeled Lys-containing peptides penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC-labeled Arg-containing peptides did not penetrate but associated with the membranes. Collectively, our results suggested that the ultimate target site of action of Arg-containing peptides and Lys-containing peptides may be the membrane and the cytoplasm of C. albicans, respectively.